Bacterial Cultures – Withering Syndrome Clones

Inoculated 5mL of LB + Amp (100ug/mL) in 50mL conicals with one of the following Withering Syndrome clones frozen stocks (located in -80C box called “WS RLP Plasmids”):

  • PWC8 WFS-RLP pCRII (from 4/13/01)
  • p16RK3 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
  • p16RK7 rickettsial 16s rDNA pCR2.1 TOPO (from 4/2/98)
  • p18RK7 WFS-RLP rDNA pCR2.1 (from 4/15/01)

Incubated O/N, 37C, 200RPM. Will isolate plasmid DNA tomorrow.

Bacteria Cultures – p16RK3-A, B, & C Clones from 5/2004

We identified a usable clone for the RLP qPCR assay that contains the entire GenBank sequence (AF133090), instead of the partial (and incorrect) clone that Lisa and I had used previously (see 20120323); leading to a month’s worth of qPCRs that failed to produce the expected, and desired, results. This clone is vector pCR2.1 (Invitrogen) containing the full GenBank sequence, AF133090).

Since the p16RK3-C clone failed to grow yesterday, decided to make cultures from all three frozen stocks of p16RK3 to ensure that at least one will grow.

Three 5mL O/N cultures in LB+Amp (100ug/mL) were inoculated directly from the frozen stocks and grown at 37C on a rocker in a 15mL conical tube.

Results:

All three cultures failed to grow. Left cultures in incubator on rocker. Will make some LB and LB+Amp plates and try streaking out all three frozen stocks. Also will try growing the frozen stocks in liquid LB (no Amp) O/N in hopes of getting a culture for mini prep.

Bacteria Culture – p16RK3-C RLP Clone from 5/2004

We identified a usable clone for the RLP qPCR assay that contains the entire GenBank sequence (AF133090), instead of the partial (and incorrect) clone that Lisa and I had used previously (see 20120323); leading to a month’s worth of qPCRs that failed to produce the expected, and desired, results. This clone is vector pCR2.1 (Invitrogen) containing the full GenBank sequence, AF133090).

A 5mL O/N culture in LB+Amp (100ug/mL) was inoculated directly from the frozen stock and grown at 37C on a rocker in a 15mL conical tube.

Results:

Culture failed to grow. Will try again, but will inoculate multiple cultures from multiple stocks to ensure that at least one will grow up. Left culture on rocker in incubator.